Glycosylation is a post-translational modification of proteins in cells. Glycans are linked to amino acids through glycosidic bonds. N-type linkages to asparagine and O-type linkages to serine and threonine are predominant forms. Protein glycosylation is involved in cellular activities, such as protein folding, immune response, and cell to cell communication. Abnormal glycosylation can play a role in cancer cell growth and metastasis.
Glycosylation sites and glycan structures can be determined in part by enrichment of glycopeptides, or by enzymatically or chemically releasing glycans. The structure of released glycans and remaining peptides can be determined by mass spectrometry and liquid chromatography/mass spectrometry. Fragmentation techniques can also be used to obtain glycan structures and amino acid sequences of the peptide backbone of glycopeptides with mass spectrometry. For example, glycosidic linkage obtained from collision induced dissociation may be combined with peptide sequence from electron-transfer dissociation to identify glycosylation sites and glycan structures of glycoproteins.
A drawback of these methods is low fragmentation efficiency of glycopeptides. Because of the structural complexity of glycans in glycopeptides, fragmentation techniques can be inefficient and time-consuming due to the need for manual data analysis from multiple experiments.
Further, a mass spectrometer for such analysis should have the ability, in a single result, to provide sequential collision-induced mass spectral analysis, so that multiple experiments would not be needed.
Moreover, a mass spectrometer for such analysis should have the ability to provide uniform collisional energies for glycopeptide fragmentation, regardless of glycopeptide structure, so that the analysis can be applied to a wide range of structures.
What is needed are methods and apparatus for accurate and efficient analysis of glycoproteins by mass spectrometry.
There is a continuing need for a mass spectrometer apparatus for analysis of glycopeptides, in a single mass spectrometric result, for identifying glycosylation sites, determining amino acid sequences, and other features.